Knockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function

Author:

Bekier Michael E.1,Wang Leibin1,Li Jie1,Huang Haoran1,Tang Danming1,Zhang Xiaoyan1,Wang Yanzhuang12

Affiliation:

1. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048

2. Department of Neurology, School of Medicine, University of Michigan, Ann Arbor, MI 48109

Abstract

Golgi reassembly stacking protein of 65 kDa (GRASP65) and Golgi reassembly stacking protein of 55 kDa (GRASP55) were originally identified as Golgi stacking proteins; however, subsequent GRASP knockdown experiments yielded inconsistent results with respect to the Golgi structure, indicating a limitation of RNAi-based depletion. In this study, we have applied the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to knock out GRASP55 and GRASP65, individually or in combination, in HeLa and HEK293 cells. We show that double knockout of GRASP proteins disperses the Golgi stack into single cisternae and tubulovesicular structures, accelerates protein trafficking, and impairs accurate glycosylation of proteins and lipids. These results demonstrate a critical role for GRASPs in maintaining the stacked structure of the Golgi, which is required for accurate posttranslational modifications in the Golgi. Additionally, the GRASP knockout cell lines developed in this study will be useful tools for studying the role of GRASP proteins in other important cellular processes.

Publisher

American Society for Cell Biology (ASCB)

Subject

Cell Biology,Molecular Biology

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